Binding proteins inhibiting the vegf-a receptor interaction

ABSTRACT

The present invention relates to binding proteins specific for VEGF-A, in particular to recombinant binding proteins comprising a binding domain, which inhibits VEGF-Axxx binding to VEGFR-2. Examples of such binding proteins are proteins which comprise an ankyrin repeat domain with the desired binding specificity. The binding proteins are useful in the treatment of cancer and other pathological conditions, e.g. eye diseases such as age-related macular degeneration.

FIELD OF THE INVENTION

The present invention relates to recombinant binding proteins specificfor VEGF-A, as well as nucleic acids encoding such VEGF-A bindingproteins, pharmaceutical compositions comprising such proteins, and theuse of such proteins in the treatment of tumors and eye diseases.

BACKGROUND OF THE INVENTION

Angiogenesis, the growth of new blood vessels from pre-existingvasculature, is a key process in several pathological conditions,including tumor growth and eye diseases, in particular ocularneovascularization diseases such as age-related macular degeneration(AMD) or diabetic macular edema (DME) (Carmeliet, P., Nature 438,932-936, 2005). Vascular endothelial growth factors (VEGFs) stimulateangiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR)tyrosine kinases in endothelial cells (Ferrara, N., Gerber, H. P. andLeCouter, J., Nature Med. 9, 669-676, 2003).

The mammalian VEGF family consists of five glycoproteins referred to asVEGF-A, VEGF-B, VEGF-C, VEGF-D (also known as FIGF) and placenta growthfactor (PIGF, also known as PGF). VEGF-A has been shown to be aneffective target for anti-angiogenic therapy (Ellis, L. M. and Hicklin,D. J., Nature Rev. Cancer 8, 579-591, 2008). The VEGF-A ligands bind toand activate three structurally similar type III receptor tyrosinekinases, designated VEGFR-1 (also known as FLT1), VEGFR-2 (also known asKDR) and VEGFR-3 (also known as FLT4). The VEGF ligands have distinctivebinding specificities for each of these tyrosine kinase receptors, whichcontribute to their diversity of function. In response to ligandbinding, the VEGFR tyrosine kinases activate a network of distinctdownstream signaling pathways. VEGFR-1 and VEGFR-2 are primarily foundon the vascular endothelium whereas VEGFR-3 is mostly found on thelymphatic endothelium.

These receptors all have an extracellular domain, a single transmembraneregion and a consensus tyrosine kinase sequence interrupted by akinase-insert domain. More recently neuropilin (NRP-1), originallyidentified as a receptor for the semaphorin/collapsin family of neuronalguidance mediators, was shown to act as an isoform specific receptor forVEGF-A.

Various isoforms of VEGF-A are known that are generated by alternativesplicing from eight exons within the VEGF-A gene. All isoforms containexons 1-5 and the terminal exon, exon 8. Exons 6 and 7, which encodeheparin-binding domains, can be included or excluded. This gives rise toa family of proteins termed according to their amino acid number:VEGF-A165, VEGF-A121, VEGF-A189, and so on. Exon 8, however, containstwo 3′ splice sites in the nucleotide sequences, which can be used bythe cell to generate two families of isoforms with identical length, butdiffering C-terminal amino acid sequences (Varey, A. H. R. et al.,British J. Cancer 98, 1366-1379, 2008). VEGF-Axxx (“xxx” denotes theamino acid number of the mature protein), the pro-angiogenic family ofisoforms, is generated by use of the most proximal sequence in exon 8(resulting in the inclusion of exon 8a). The more recently describedanti-angiogenic VEGF-Axxxb isoforms are generated by the use of a distalsplice site, 66 by further along the gene from the proximal splice site.This results in splicing out of exon 8a and the production of mRNAsequences that encode the VEGF-Axxxb family. VEGF-A165 is thepredominant pro-angiogenic isoform and is commonly overexpressed in avariety of human solid tumors. VEGF-A165b was the first of the exon8b-encoded isoforms identified and was shown to have anti-angiogeniceffects (Varey et al., loc. cit.; Konopatskaya, O. et al., MolecularVision 12, 626-632, 2006). It is an endogenous inhibitory form ofVEGF-A, which decreases VEGF-A induced proliferation and migration ofendothelial cells. Although it can bind to VEGFR-2, VEGF-A165b bindingdoes not result in receptor phosphorylation or activation of thedownstream signaling pathways.

There are several approaches to inhibiting VEGF-A signaling, includingneutralization of the ligand or receptor by antibodies, and blockingVEGF-A receptor activation and signaling with tyrosine kinaseinhibitors. VEGF-A targeted therapy has been shown to be efficacious asa single agent in AMD, DME, renal cell carcinoma and hepatocellularcarcinoma, whereas it is only of benefit when combined with chemotherapyfor patients with metastatic colorectal, non-small-cell lung andmetastatic breast cancer (Narayanan, R. et al., Nat Rev. Drug Discov. 5,815-816, 2005; Ellis and Hicklin, loc. cit).

Beside antibodies other binding domains can be used to neutralize aligand or a receptor (Skerra, A., J. Mol. Recog. 13, 167-187, 2000;Binz, H. K., Amstutz, P. and Plückthun, A., Nat. Biotechnol. 23,1257-1268, 2005). One such novel class of binding domains are based ondesigned repeat domains (WO 02/20565; Binz, H. K., Amstutz, P., Kohl,A., Stumpp, M. T., Briand, C., Forrer, P., Grütter, M. G., andPlückthun, A., Nat. Biotechnol. 22, 575-582, 2004). WO 02/20565describes how large libraries of repeat proteins can be constructed andtheir general application. Nevertheless, WO 02/20565 does neitherdisclose the selection of repeat domains with binding specificity forVEGF-Axxx nor concrete repeat sequence motifs of repeat domains thatspecifically bind to VEGF-Axxx.

Targeting VEGF-A with currently available therapeutics is not effectivein all patients, or for all diseases (e.g., EGFR-expressing cancers). Ithas even become increasingly apparent that the therapeutic benefitassociated with VEGF-A targeted therapy is complex and probably involvesmultiple mechanisms (Ellis and Hicklin, loc. cit.). For example,marketed anti-VEGF drugs, such as bevacizumab (Avastin®) or ranibizumab(Lucentis®) (see WO 96/030046, WO 98/045331 and WO 98/045332) or drugsin clinical development, such as VEGF-Trap® (WO 00/075319) do notdistinguish between the pro- and anti-angiogenic forms of VEGF-A, sothey do inhibit both. As a result, they inhibit angiogenesis, but alsodeprive healthy tissues of an essential survival factor, namelyVEGF-Axxxb, resulting in cytotoxicity and dose-limiting side effects,which in turn limit efficacy. Side effects common to current anti-VEGF-Atherapies are gastrointestinal perforations, bleeding, hypertension,thromboembolic events and proteinuria (Kamba, T. and McDonald, D. M.,Br. J. Cancer 96, 1788-95, 2007). Thus, a need exists for improvedanti-angiogenic agents for treating cancer and other pathologicalconditions.

The technical problem underlying the present invention is to identifynovel anti-angiogenic agents, such as repeat domains with bindingspecificity to VEGF-Axxx, for an improved treatment of cancer and otherpathological conditions, e.g. eye diseases such as AMD or DME. Thesolution to this technical problem is achieved by providing theembodiments characterized in the claims.

SUMMARY OF THE INVENTION

The present invention relates to a binding protein comprising a bindingdomain, wherein said binding domain inhibits VEGF-Axxx binding toVEGFR-2 and wherein said binding domain has a midpoint denaturationtemperature (Tm) above 40° C. upon thermal unfolding and forms less than5% (w/w) insoluble aggregates at concentrations up to 10 g/L whenincubated at 37° C. for 1 day in PBS. More specifically the inventionrelates to a recombinant binding protein comprising at least one repeatdomain, wherein said repeat domain binds VEGF-Axxx with a Kd below 10⁻⁷Mand inhibits VEGF-Axxx binding to VEGFR-2. In particular such a bindingprotein inhibits sprouting of HUVEC spheroids with an IC₅₀ value below10 nM, and such a binding protein has a dissociation constant K_(d) forthe interaction with VEGF-Axxxb that is at least 10-fold higher comparedto its K_(d) for the interaction with VEGF-Axxx.

In particular, the invention relates to a recombinant binding proteincomprising a binding domain with specificity for VEGF-A, which is arepeat domain, for example an ankyrin repeat domain, in particular anankyrin repeat domain comprising a repeat module with the ankyrin repeatsequence motif

(SEQ ID NO: 1) 1D23G4TPLHLAA56GHLEIVEVLLK7GADVNA wherein 1, 2, 3, 4, 5, 6, and 7, represent, independently of each other,an amino acid residue selected from the group consisting of A, D, E, F,H, I, K, L, M, N, Q, R, S, T, V, W and Y.

The invention also relates to a recombinant binding protein comprising arepeat domain with binding specificity for VEGF-A, which has at least70% amino acid sequence identity with an ankyrin repeat domain of thepresent invention, or which comprises a repeat module with at least 70%amino acid sequence identity with an ankyrin repeat module of thepresent invention, or wherein one or more of the amino acid residues ofthe ankyrin repeat modules are exchanged by an amino acid residue foundat the corresponding position on alignment of an ankyrin repeat unit.

The invention further relates to binding proteins comprising arecombinant binding protein of the present invention bound to one ormore additional moieties, for example, a moiety that also binds toVEGFR-2 or to a different target, a labeling moiety, a moiety thatfacilitates protein purification, or a moiety that provides improvedpharmacokinetics, for example a polyethylene glycol moiety. In certainembodiments, the additional moiety is a proteinaceous moiety. In certainother embodiments, the additional moiety is a non-proteinaceous polymermoiety.

The invention further relates to nucleic acid molecules encoding therecombinant binding proteins of the present invention, and to apharmaceutical composition comprising one or more of the above mentionedbinding proteins or nucleic acid molecules.

The invention further relates to a method of treatment of cancer andother pathological conditions, e.g. eye diseases such as AMD or DME,using the binding proteins of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Specific dog VEGF-A164 binding of selected designed ankyrinrepeat proteins. The interaction of selected clones with dog VEGF-A164(VEGF) and a negative control protein (MBP, E. coli maltose bindingprotein) is shown by crude extract ELISA. The biotinylated dog VEGF-A164and MBP were immobilized over NeutrAvidin. The numbers refer to singleDARPin clones selected in ribosome display against dog VEGF-A164 or thecorresponding human VEGF-A165. A=Absorbance. White bars indicate bindingto dog VEGF-A164, black bars show non-specific background binding toMBP.

FIGS. 2A and 2B. Spheroid outgrowth inhibition by a selected DARPin.FIG. 2A shows the length of sprouts in a spheroid outgrowth inhibitionassay in the presence of various concentrations of DARPin #30 (SEQ IDNO:29), a DARPin with specificity to VEGF-Axxx. FIG. 2B shows the lengthof sprouts in a spheroid outgrowth inhibition assay in the presence ofvarious concentrations of DARPin NC, a negative control DARPin with nospecificity for VEGF-Axxx.

FIGS. 3A, 3B, 3C, and 3D. Specific recognition of VEGF-A isoforms.Surface Plasmon Resonance (SPR) analysis of binding proteins on VEGF-Aisoforms. FIG. 3A shows the SPR analysis of Avastin® where 250 nM ofAvastin® was applied to a flow cell with immobilized dog VEGF-A164 for100 seconds, followed by washing with buffer flow. FIG. 3B shows the SPRanalysis of Avastin® where 250 nM of Avastin® was applied to a flow cellwith immobilized dog VEGF-A164b for 100 seconds, followed by washingwith buffer flow.

FIG. 3C shows the SPR analysis of DARPin #27 (SEQ ID NO:16) where 250 nMof DARPin #27 was applied to a flow cell with immobilized dog VEGF-A164for 100 seconds, followed by washing with buffer flow. FIG. 3D shows theSPR analysis of DARPin #27 (SEQ ID NO:16) where 250 nM of DARPin #27 wasapplied to a flow cell with immobilized dog VEGF-A164b for 100 seconds,followed by washing with buffer flow. RU=Resonance Units.

FIG. 4. Efficient inhibition of human VEGF-A165 in the rabbit eye.Vascular leakage rabbit model to show the efficacy of a DARPin ininhibiting human VEGF-A165 in the eye in comparison to Lucentis®. At day1 either PBS, DARPin #30 or Lucentis® is applied by an intravitrealinjection into one eye of each rabbit (treated eye).

At day 4 or day 30 both eyes of each rabbit were challenged byintravitreal injection of 500 ng of human VEGF-A165. All eyes wereevaluated 48 hours after the VEGF-A165 injection by measuring thefluorescein content in the vitreous and retina of all eyes one hourafter intravenous injection of sodium fluorescein.

R=ratio of fluorescein measurements treated eye/untreated eye. Standarddeviations are shown by an error bar. 4-PBS=ratio 4 days after injectionof PBS (control); 4-D=ratio 4 days after injection of DARPin #30;30-D=ratio 30 days after injection of DARPin #30; 4-L=ratio 4 days afterinjection of Lucentis®; 30-L=ratio 30 days after injection of Lucentis®.

DETAILED DESCRIPTION OF THE INVENTION

Mammalian VEGF-A exists as two families of alternative spliced isoforms:(i) the pro-angiogenic “VEGF-Axxx” isoforms generated by proximalsplicing of exon 8 and (ii) the anti-angiogenic “VEGF-Axxxb” isoformsgenerated by distal splicing of exon 8. Preferably, the binding domainaccording to the invention is specific for the pro-angiogenic VEGF-Axxxof dog, rabbit, monkey or human origin. More preferably, the bindingdomain according to the invention is specific for the pro-angiogenicVEGF-Axxx of human origin. Most preferred, the binding domain accordingto the invention is specific for human VEGF-A165. The term “protein”refers to a polypeptide, wherein at least part of the polypeptide has,or is able to, acquire a defined three-dimensional arrangement byforming secondary, tertiary, or quaternary structures within and/orbetween its polypeptide chain(s). If a protein comprises two or morepolypeptides, the individual polypeptide chains may be linkednon-covalently or covalently, e.g. by a disulfide bond between twopolypeptides. A part of a protein, which individually has, or is able toacquire a defined three-dimensional arrangement by forming secondary ortertiary structures, is termed “protein domain”. Such protein domainsare well known to the practitioner skilled in the art.

The term “recombinant” as used in recombinant protein, recombinantprotein domain and the like, means that said polypeptides are producedby the use of recombinant DNA technologies well known by thepractitioner skilled in the relevant art. For example, a recombinant DNAmolecule (e.g. produced by gene synthesis) encoding a polypeptide can becloned into a bacterial expression plasmid (e.g. pQE30, Qiagen). Whensuch a constructed recombinant expression plasmid is inserted into abacteria (e.g. E. coli), this bacteria can produce the polypeptideencoded by this recombinant DNA. The correspondingly producedpolypeptide is called a recombinant polypeptide.

The term “polypeptide tag” refers to an amino acid sequence attached toa polypeptide/protein, wherein said amino acid sequence is useful forthe purification, detection, or targeting of said polypeptide/protein,or wherein said amino acid sequence improves the physicochemicalbehavior of the polypeptide/protein, or wherein said amino acid sequencepossesses an effector function. The individual polypeptide tags,moieties and/or domains of a binding protein may be connected to eachother directly or via polypeptide linkers. These polypeptide tags areall well known in the art and are fully available to the person skilledin the art. Examples of polypeptide tags are small polypeptidesequences, for example, His, myc, FLAG, or Strep-tags or moieties suchas enzymes (for example enzymes like alkaline phosphatase), which allowthe detection of said polypeptide/protein, or moieties which can be usedfor targeting (such as immunoglobulins or fragments thereof) and/or aseffector molecules.

The term “polypeptide linker” refers to an amino acid sequence, which isable to link, for example, two protein domains, a polypeptide tag and aprotein domain, a protein domain and a non-polypeptide moiety such aspolyethylene glycol or two sequence tags. Such additional domains, tags,non-polypeptide moieties and linkers are known to the person skilled inthe relevant art. A list of example is provided in the description ofthe patent application WO 02/20565. Particular examples of such linkersare glycine-serine-linkers of variable lengths; preferably, said linkershave a length between 2 and 16 amino acids.

In the context of the present invention, the term “polypeptide” relatesto a molecule consisting of one or more chains of multiple, i.e. two ormore, amino acids linked via peptide bonds. Preferably, a polypeptideconsists of more than eight amino acids linked via peptide bonds.

The term “binding protein” refers to a protein comprising one or morebinding domains as further explained below. Preferably, said bindingprotein comprises up to four binding domains. More preferably, saidbinding protein comprises up to two binding domains. Most preferably,said binding protein comprises only one binding domain. Furthermore, anysuch binding protein may comprise additional protein domains that arenot binding domains, multimerization moieties, polypeptide tags,polypeptide linkers and/or non-proteinaceous polymer molecules. Examplesof multimerization moieties are immunoglobulin heavy chain constantregions which pair to provide functional immunoglobulin Fc domains, andleucine zippers or polypeptides comprising a free thiol which forms anintermolecular disulfide bond between two such polypeptides. Examples ofnon-proteinaceous polymer molecules are hydroxyethyl starch (HES),polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene.

The term “PEGylated” means that a PEG moiety is covalently attached to,for example, a polypeptide of the invention.

The term “binding domain” means a protein domain exhibiting the same“fold” (three-dimensional arrangement) as a protein scaffold and havinga predetermined property, as defined below. Such a binding domain may beobtained by rational, or most commonly, combinatorial proteinengineering techniques, skills which are known in the art (Skerra, 2000,loc. cit.; Binz et al., 2005, loc. cit.). For example, a binding domainhaving a predetermined property can be obtained by a method comprisingthe steps of (a) providing a diverse collection of protein domainsexhibiting the same fold as a protein scaffold as defined further below;and (b) screening said diverse collection and/or selecting from saiddiverse collection to obtain at least one protein domain having saidpredetermined property. The diverse collection of protein domains may beprovided by several methods in accordance with the screening and/orselection system being used, and may comprise the use of methods wellknown to the person skilled in the art, such as phage display orribosome display.

The term “protein scaffold” means a protein with exposed surface areasin which amino acid insertions, substitutions or deletions are highlytolerable. Examples of protein scaffolds that can be used to generatebinding domains of the present invention are antibodies or fragmentsthereof such as single-chain Fv or Fab fragments, protein A fromStaphylococcus aureus, the bilin binding protein from Pieris brassicaeor other lipocalins, ankyrin repeat proteins or other repeat proteins,and human fibronectin. Protein scaffolds are known to the person skilledin the art (Binz et al., 2005, loc. cit.; Binz et al., 2004, loc. cit.).

The term “predetermined property” refers to a property such as bindingto a target, blocking of a target, activation of a target-mediatedreaction, enzymatic activity, and related further properties. Dependingon the type of desired property, one of ordinary skill will be able toidentify format and necessary steps for performing screening and/orselection of a binding domain with the desired property. Preferably,said predetermined property is binding to a target.

Preferably, the binding protein of the invention is not an antibody or afragment thereof, such as Fab or scFv fragments. Antibodies andfragments thereof are well known to the person skilled in the art.

Also preferably, the binding domain of the invention does not comprisean immunoglobulin fold as present in antibodies and/or the fibronectintype III domain. An immunoglobulin fold is a common all-β protein foldthat consists of a 2-layer sandwich of about 7 anti-parallel β-strandsarranged in two β-sheets. Immunoglobulin folds are well known to theperson skilled in the art. For example, such binding domains comprisingan immunoglobulin fold are described in WO 07/080392 or WO 08/097497.

Further preferably, the binding domain of the invention does notcomprise an immunoglobulin-like domain as found in VEGFR-1 or VEGFR-2.Such binding domains are described in WO 00/075319.

A preferred binding domain is a binding domain having anti-angiogeniceffects. The anti-angiogenic effect of a binding domain can bedetermined by assays well know to the person skilled in the art, such asthe sprouting assay of HUVEC spheroids described in Example 2.

Further preferred is a binding domain comprising between 70 and 300amino acids, in particular between 100 and 200 amino acids.

Further preferred is a binding domain devoid of a free Cys residue. Afree Cys residue is not involved in the formation of a disulfide bond.Even more preferred is a binding domain free of any Cys residue.

A preferred binding domain of the invention is a repeat domain or adesigned repeat domain, preferably as described in WO 02/20565.

A particularly preferred binding domain is a designed ankyrin repeatdomain (Binz, H. K. et al., 2004, loc. cit.), preferably as described inWO 02/20565. Examples of designed ankyrin repeat domains are shown inthe Examples.

The definitions hereinafter for repeat proteins are based on those inpatent application WO 02/20565. Patent application WO 02/20565 furthercontains a general description of repeat protein features, techniquesand applications.

The term “repeat proteins” refers to a protein comprising one or morerepeat domains. Preferably, each of said repeat proteins comprises up tofour repeat domains. More preferably, each of said repeat proteinscomprises up to two repeat domains. Most preferably, each of the repeatproteins comprises only one repeat domain. Furthermore, said repeatprotein may comprise additional non-repeat protein domains, polypeptidetags and/or polypeptide linkers.

The term “repeat domain” refers to a protein domain comprising two ormore consecutive repeat units (modules) as structural units, whereinsaid structural units have the same fold, and stack tightly to create,for example, a superhelical structure having a joint hydrophobic core.

The term “designed repeat protein” and “designed repeat domain” refer toa repeat protein or repeat domain, respectively, obtained as the resultof the inventive procedure explained in patent application WO 02/20565.Designed repeat proteins and designed repeat domains are synthetic andnot from nature. They are man-made proteins or domains, respectively,obtained by expression of correspondingly designed nucleic acids.Preferably, the expression is done in eukaryotic or prokaryotic cells,such as bacterial cells, or by using a cell-free in vitro expressionsystem.

The term “structural unit” refers to a locally ordered part of apolypeptide, formed by three-dimensional interactions between two ormore segments of secondary structure that are near one another along thepolypeptide chain. Such a structural unit exhibits a structural motif.The term “structural motif” refers to a three-dimensional arrangement ofsecondary structure elements present in at least one structural unit.Structural motifs are well known to the person skilled in the art.Structural units alone are not able to acquire a definedthree-dimensional arrangement; however, their consecutive arrangement,for example as repeat modules in a repeat domain, leads to a mutualstabilization of neighboring units resulting in a superhelicalstructure.

The term “repeat unit” refers to amino acid sequences comprising repeatsequence motifs of one or more naturally occurring repeat proteins,wherein said “repeat units” are found in multiple copies, and whichexhibit a defined folding topology common to all said motifs determiningthe fold of the protein. Such repeat units comprise framework residuesand interaction residues. Examples of such repeat units are armadillorepeat units, leucine-rich repeat units, ankyrin repeat units,tetratricopeptide repeat units, HEAT repeat units, and leucine-richvariant repeat units. Naturally occurring proteins containing two ormore such repeat units are referred to as “naturally occurring repeatproteins”. The amino acid sequences of the individual repeat units of arepeat protein may have a significant number of mutations,substitutions, additions and/or deletions when compared to each other,while still substantially retaining the general pattern, or motif, ofthe repeat units.

The term “framework residues” relates to amino acid residues of therepeat units, or the corresponding amino acid residues of the repeatmodules, which contribute to the folding topology, i.e. which contributeto the fold of said repeat unit (or module) or which contribute to theinteraction with a neighboring unit (or module). Such contribution mightbe the interaction with other residues in the repeat unit (module), orthe influence on the polypeptide backbone conformation as found inα-helices or β-sheets, or amino acid stretches forming linearpolypeptides or loops.

The term “target interaction residues” refers to amino acid residues ofthe repeat units, or the corresponding amino acid residues of the repeatmodules, which contribute to the interaction with target substances.Such contribution might be the direct interaction with the targetsubstances, or the influence on other directly interacting residues,e.g. by stabilizing the conformation of the polypeptide of a repeat unit(module) to allow or enhance the interaction of directly interactingresidues with said target. Such framework and target interactionresidues may be identified by analysis of the structural data obtainedby physicochemical methods, such as X-ray crystallography, NMR and/or CDspectroscopy, or by comparison with known and related structuralinformation well known to practitioners in structural biology and/orbioinformatics.

Preferably, the repeat units used for the deduction of a repeat sequencemotif are homologous repeat units, wherein the repeat units comprise thesame structural motif and wherein more than 70% of the frameworkresidues of said repeat units are homologous to each other. Preferably,more than 80% of the framework residues of said repeat units arehomologous. Most preferably, more than 90% of the framework residues ofsaid repeat units are homologous. Computer programs to determine thepercentage of homology between polypeptides, such as Fasta, Blast orGap, are known to the person skilled in the art. Further preferably, therepeat units used for the deduction of a repeat sequence motif arehomologous repeat units obtained from repeat domains selected on atarget, for example as described in Example 1 and having the sametarget-specificity.

The term “repeat sequence motif” refers to an amino acid sequence, whichis deduced from one or more repeat units. Preferably, said repeat unitsare from repeat domains having binding specificity for the same target.Such repeat sequence motifs comprise framework residue positions andtarget interaction residue positions. Said framework residue positionscorrespond to the positions of framework residues of the repeat units.Likewise, said target interaction residue positions correspond to thepositions of target interaction residues of the repeat units. Repeatsequence motifs comprise fixed positions and randomized positions. Theterm “fixed position” refers to an amino acid position in a repeatsequence motif, wherein said position is set to a particular amino acid.Most often, such fixed positions correspond to the positions offramework residues and/or the positions of target interaction residuesthat are specific for a certain target. The term “randomized position”refers to an amino acid position in a repeat sequence motif, wherein twoor more amino acids are allowed at said amino acid position, forexample, wherein any of the usual twenty naturally occurring amino acidsare allowed, or wherein most of the twenty naturally occurring aminoacids are allowed, such as amino acids other than cysteine, or aminoacids other than glycine, cysteine and proline. Most often, suchrandomized positions correspond to the positions of target interactionresidues. However, some positions of framework residues may also berandomized.

The term “folding topology” refers to the tertiary structure of saidrepeat units. The folding topology will be determined by stretches ofamino acids forming at least parts of α-helices or β-sheets, or aminoacid stretches forming linear polypeptides or loops, or any combinationof α-helices, β-sheets and/or linear polypeptides/loops.

The term “consecutive” refers to an arrangement, wherein the repeatunits or repeat modules are arranged in tandem. In designed repeatproteins, there are at least 2, usually about 2 to 6, in particular atleast about 6, frequently 20 or more repeat units. In most cases, repeatunits will exhibit a high degree of sequence identity (same amino acidresidues at corresponding positions) or sequence similarity (amino acidresidues being different, but having similar physicochemicalproperties), and some of the amino acid residues might be key residuesbeing strongly conserved in the different repeat units found innaturally occurring proteins. However, a high degree of sequencevariability by amino acid insertions and/or deletions, and/orsubstitutions between the different repeat units found in naturallyoccurring proteins will be possible as long as the common foldingtopology is maintained.

Methods for directly determining the folding topology of repeat proteinsby physico-chemical means such as X-ray crystallography, NMR or CDspectroscopy, are well known to the practitioner skilled in the art.Methods for identifying and determining repeat units or repeat sequencemotifs or for identifying families of related proteins comprising suchrepeat units or motifs, such as homology searches (BLAST etc.), are wellestablished in the field of bioinformatics, and are well known to thepractitioner in the art. The step of refining an initial repeat sequencemotif may comprise an iterative process.

The term “repeat modules” refers to the repeated amino acid sequences ofthe designed repeat domains, which are originally derived from therepeat units of naturally occurring repeat proteins. Each repeat modulecomprised in a repeat domain is derived from one or more repeat units ofthe family or subfamily of naturally occurring repeat proteins, e.g. thefamily of armadillo repeat proteins or ankyrin repeat proteins.

“Repeat modules” may comprise positions with amino acid residues presentin all copies of corresponding repeat modules (“fixed positions”) andpositions with differing or “randomized” amino acid residues(“randomized positions”).

The term “capping module” refers to a polypeptide fused to the N- orC-terminal repeat module of a repeat domain, wherein said capping moduleforms tight tertiary interactions with said repeat module therebyproviding a cap that shields the hydrophobic core of said repeat moduleat the side not in contact with the consecutive repeat module from thesolvent. Said N- and/or C-terminal capping module may be, or may bederived from, a capping unit or other domain found in a naturallyoccurring repeat protein adjacent to a repeat unit. The term “cappingunit” refers to a naturally occurring folded polypeptide, wherein saidpolypeptide defines a particular structural unit which is N- orC-terminally fused to a repeat unit, wherein said polypeptide formstight tertiary interactions with said repeat unit thereby providing acap that shields the hydrophobic core of said repeat unit at one sidefrom the solvent. Such capping units may have sequence similarities tosaid repeat sequence motif. Capping modules and capping repeats aredescribed in WO 02/020565. For example, the N-terminal capping module ofSEQ ID NO:21 is encoded by the amino acids from position 1 to 32. Alsopreferred is such an N-terminal capping module having a glycine oraspartate residue at position 5.

The term “target” refers to an individual molecule such as a nucleicacid molecule, a polypeptide or protein, a carbohydrate, or any othernaturally occurring molecule, including any part of such individualmolecule, or complexes of two or more of such molecules. The target maybe a whole cell or a tissue sample, or it may be any non-naturalmolecule or moiety. Preferably, the target is a naturally occurring ornon-natural polypeptide or a polypeptide containing chemicalmodifications, for example modified by natural or non-naturalphosphorylation, acetylation, or methylation. In the particularapplication of the present invention, the target is VEGF-Axxx orVEGFR-2.

The term “consensus sequence” refers to an amino acid sequence, whereinsaid consensus sequence is obtained by structural and/or sequencealigning of multiple repeat units. Using two or more structural and/orsequence aligned repeat units, and allowing for gaps in the alignment,it is possible to determine the most frequent amino acid residue at eachposition. The consensus sequence is that sequence which comprises theamino acids which are most frequently represented at each position. Inthe event that two or more amino acids are represented above-average ata single position, the consensus sequence may include a subset of thoseamino acids. Said two or more repeat units may be taken from the repeatunits comprised in a single repeat protein, or from two or moredifferent repeat proteins.

Consensus sequences and methods to determine them are well known to theperson skilled in the art.

A “consensus amino acid residue” is the amino acid found at a certainposition in a consensus sequence. If two or more, e.g. three, four orfive, amino acid residues are found with a similar probability in saidtwo or more repeat units, the consensus amino acid may be one of themost frequently found amino acids or a combination of said two or moreamino acid residues.

Further preferred are non-naturally occurring binding proteins orbinding domains.

The term “non-naturally occurring” means synthetic or not from nature,more specifically, the term means made from the hand of man. The term“non-naturally occurring binding protein” or “non-naturally occurringbinding domain” means that said binding protein or said binding domainis synthetic (i.e. produced by chemical synthesis from amino acids) orrecombinant and not from nature. “Non-naturally occurring bindingprotein” or “non-naturally occurring binding domain” is a man-madeprotein or domain, respectively, obtained by expression ofcorrespondingly designed nucleic acids. Preferably, the expression isdone in eukaryotic or bacterial cells, or by using a cell-free in vitroexpression system. Further, the term means that the sequence of saidbinding protein or said binding domain is not present as anon-artificial sequence entry in a sequence database, for example inGenBank, EMBL-Bank or Swiss-Prot. These databases and other similarsequence databases are well known to the person skilled in the art.

The invention relates to a binding protein comprising a binding domain,wherein said binding domain inhibits VEGF-Axxx binding to VEGFR-2 andwherein said binding protein and/or binding domain has a midpointdenaturation temperature (Tm) above 40° C. upon thermal unfolding andforms less than 5% (w/w) insoluble aggregates at concentrations up to 10g/L when incubated at 37° C. for 1 day in phosphate buffered saline(PBS).

A binding domain can inhibit VEGF-Axxx binding to VEGFR-2 either bybinding to VEGF-Axxx or by binding to VEGFR-2 in a way that the apparentdissociation constant (K_(d)) between VEGF-Axxx and VEGFR-2 is increasedmore than 10²-fold, preferably more than 10³-fold, more preferably morethan 10⁴-fold, more preferably more than 10⁵-fold, and most preferablymore than 10⁶-fold. Preferably, the K_(d) for the interaction of thebinding domain to either VEGF-Axxx or VEGFR-2 is below 10⁻⁷M, preferablybelow 10⁻⁸M, more preferably below 10⁻⁶M, more preferably below 10⁻¹⁰M,and most preferably below 10⁻¹¹M. Methods, to determine dissociationconstants of protein-protein interactions, such as surface plasmonresonance (SPR) based technologies, are well known to the person skilledin the art.

A preferred binding domain binds VEGF-Axxx. Even more preferred is abinding domain that binds human VEGF-A165.

The term “PBS” means a phosphate buffered water solution containing 137mM NaCl, 10 mM phosphate and 2.7 mM KCl and having a pH of 7.4.

Preferably, the binding protein and/or binding domain has a midpointdenaturation temperature (Tm) above 45° C., more preferably above 50°C., more preferably above 55° C., and most preferably above 60° C. uponthermal unfolding. A binding protein or a binding domain of theinvention possesses a defined secondary and tertiary structure underphysiological conditions. Thermal unfolding of such a polypeptideresults in a loss of its tertiary and secondary structure, which can befollowed, for example, by circular dichroism (CD) measurements. Themidpoint denaturation temperature of a binding protein or binding domainupon thermal unfolding corresponds to the temperature at the midpoint ofthe cooperative transition in physiological buffer upon heatdenaturation of said protein or domain by slowly increasing thetemperature from 10° C. to about 100° C. The determination of a midpointdenaturation temperature upon thermal unfolding is well known to theperson skilled in the art. This midpoint denaturation temperature of abinding protein or binding domain upon thermal unfolding is indicativeof the thermal stability of said polypeptide.

Also preferred is a binding protein and/or binding domain forming lessthan 5% (w/w) insoluble aggregates at concentrations up to 20 g/l,preferably up 40 g/L, more preferably up to 60 g/L, even more preferablyup to 80 g/L, and most preferably up to 100 g/L when incubated for over5 days, preferably over 10 days, more preferably over 20 days, morepreferably over 40 days, and most preferably over 100 days at 37° C. inPBS. The formation of insoluble aggregates can be detected by theappearance of visual precipitations, gel filtration or dynamic lightscattering, which strongly increases upon formation of insolubleaggregates. Insoluble aggregates can be removed from a protein sample bycentrifugation at 10,000 xg for 10 minutes. Preferably, a bindingprotein and/or binding domain forms less than 2%, 1%, 0.5%, 0.2%, 0.1%,or 0.05% (w/w) insoluble aggregates under the mentioned incubationconditions at 37° C. in PBS. Percentages of insoluble aggregates can bedetermined by separation of the insoluble aggregates from solubleprotein, followed by determination of the protein amounts in the solubleand insoluble fraction by standard quantification methods.

Also preferred is a binding protein and/or binding domain that does notlose its native three-dimensional structure upon incubation in PBScontaining 100 mM dithiothreitol (DTT) for 1 or 10 hours at 37° C.

In one particular embodiment the invention relates to a binding proteincomprising a binding domain inhibiting VEGF-Axxx binding to VEGFR-2 andhaving the indicated or preferred midpoint denaturation temperature andnon-aggregating properties as defined above, wherein said bindingprotein inhibits sprouting of HUVEC spheroids with an IC₅₀ value below100 nM.

The term “HUVEC” means human umbilical vein endothelial cells, which canbe isolated from normal human umbilical vein and which are responsive toVEGF-A stimulation. Assays to measure the sprouting of HUVEC spheroids,such as that described in Example 2, are well known to the personskilled in the art.

An IC₅₀ value is the concentration of a substance, such as a bindingprotein or binding domain, which is required for 50% inhibition in vitroof an experimental determined parameter, such as the sprouting of HUVECspheroids. IC₅₀ values can be readily determined by the person skilledin the art (Korff T. and Augustin H. G., J. Cell Biol. 143(5), 1341-52,1998).

Preferred is a binding protein and/or binding domain that inhibits thesprouting of HUVEC spheroid with an IC₅₀ value below 10 nM, preferablybelow 1 nM, more preferably below 0.1 nM, and most preferably below 0.05nM.

Further preferred is a monomeric binding protein and/or binding domainthat inhibits the sprouting of HUVEC spheroids with an IC₅₀ value lowerthan the corresponding IC₅₀ value of ranibizumab (Lucentis®, aregistered trademark of Genentech), bevacizumab (Avastin®, a registeredtrademark of Genentech), aflibercept (VEGF Trap®, a registered trademarkof Regeneron), or pegaptanib (Macugen®, a registered trademark ofPfizer).

In particular the invention relates to a binding protein comprising abinding domain inhibiting VEGF-Axxx binding to VEGFR-2 and having theindicated or preferred midpoint denaturation temperature andnon-aggregating properties as defined above, wherein the K_(d) for theinteraction of said binding domain to VEGF-Axxxb is at least 10-foldhigher compared to the K_(d) for the interaction of said binding domainto the corresponding VEGF-Axxx.

Preferably, the K_(d) for the interaction of the binding domain toVEGF-Axxxb is at least 10²-fold higher, preferably 10³-fold higher, morepreferably 10⁴-fold higher, more preferably 10⁵-fold higher, and mostpreferably 10⁶-fold higher compared to the K_(d) for the interaction ofthe binding domain to the corresponding VEGF-Axxx.

Also preferably, the K_(d) for the interaction of a binding domain toVEGF-Axxxb is above 10³ nM and the K_(d) for the interaction of thebinding domain to VEGF-Axxx is below 10 or 1 nM.

The K_(d) for the interaction of a preferred binding domain to VEGF-B,VEGF-C, VEGF-D, PIGF or PDGF is above 1 nM, preferably above 10 nM, morepreferably above 10² nM, even more preferably above 10³ nM, and mostpreferably above10⁴ nM.

Preferably, VEGF-Axxx is either dog VEGF-A164 or simian VEGF-A165 orhuman VEGF-A165, and VEGF-Axxxb is either dog VEGF-A164b or simianVEGF-A165b or human VEGF-A165b.

Another preferred embodiment is a recombinant binding protein comprisinga binding domain, wherein said binding domain inhibits VEGF-Axxx bindingto VEGFR-2 and wherein said binding domain is a repeat domain or adesigned repeat domain. Such a repeat domain may comprise one, two,three or more internal repeat modules that will participate in bindingto VEGF-Axxx. Preferably, such a repeat domain comprises an N-terminalcapping module, two to four internal repeat modules, and a C-terminalcapping module. Preferably, said binding domain is an ankyrin repeatdomain or designed ankyrin repeat domain.

Preferred is a recombinant binding protein, wherein said ankyrin repeatdomain or said designed ankyrin repeat domain comprises a repeat modulewith the ankyrin repeat sequence motif

(SEQ ID NO: 1) 1D23G4TPLHLAA56GHLEIVEVLLK7GADVNA wherein 1, 2, 3, 4, 5, 6, and 7, represent, independently of each other,an amino acid residue selected from the group A, D, E, F, H, I, K, L, M,N, Q, R, S, T, V, W and Y.

Particularly preferred is a recombinant binding protein, wherein saidankyrin repeat domain or said designed ankyrin repeat domain comprises arepeat module with the ankyrin repeat sequence motif

(SEQ ID NO: 2) 1D23GWTPLHLAA45GHLEIVEVLLK6GADVNA wherein 1 represents an amino acid residue selected from the groupconsisting of F, T, N, R, V, A, I, K, Q, S and Y; preferably F, T, N, Rand V; more preferably F and T;

2 represents an amino acid residue selected from the group consisting ofW, Y, H and F; preferably W, Y and H;

3 represents an amino acid residue selected from the group consisting ofM, I, F and V; preferably M and I;

4 represents an amino acid residue selected from the group consisting ofH, A, K, G, L, M, N, T, V, W and Y; preferably H, A and K;

5 represents an amino acid residue selected from the group consisting ofE, Y, F, V, H, I, L, N and R; preferably E, Y, F, V and H; morepreferably E, Y, F and V; and 6 represents an amino acid residueselected from the group consisting of A, N, Y, H and R.

Further preferred is a recombinant binding protein, wherein said ankyrinrepeat domain or said designed ankyrin repeat domain comprises a repeatmodule with the ankyrin repeat sequence motif

(SEQ ID NO: 3) 1D23G4TPLHLAA56GHLEIVEVLLK7GADVN8 wherein

1 represents an amino acid residue selected from the group consisting ofT, E, A, D, F, K, N, Q, R, S, W and Y; preferably T and E;

2 represents an amino acid residue selected from the group consisting ofV, F, Y, A, H, I, K, R, T and W; preferably V, F and Y;

3 represents an amino acid residue selected from the group consisting ofS, A, N, F and M; preferably S, A and N; more preferably S and A;

4 represents an amino acid residue selected from the group consisting ofY, F, S and W;

5 represents an amino acid residue selected from the group consisting ofA, S, L and Y; preferably A and S;

6 represents an amino acid residue selected from the group consisting ofD, N, M, A, I, K and Y; preferably D, N and M; more preferably D and N;

7 represents an amino acid residue selected from the group consisting ofA, Y, H, N and D; and

8 represents the amino acid residue T or A.

Further preferred is a recombinant binding protein, wherein said ankyrinrepeat domain or said designed ankyrin repeat domain comprises a repeatmodule with the ankyrin repeat sequence motif

(SEQ ID NO: 4) 1D23GWTPLHL4ADLG5LEIVEVLLK6GADVN7 wherein

1 represents an amino acid residue selected from the group consisting ofK, T and Y;

2 represents the amino acid residue N or M;

3 represents the amino acid residue T or F;

4 represents the amino acid residue S or A;

5 represents the amino acid residue H or R;

6 represents an amino acid residue selected from the group consisting ofA, Y, H and N; and

7 represents the amino acid residue A or T.

Even more preferred is a recombinant binding protein, wherein saidankyrin repeat domain or said designed ankyrin repeat domain comprises arepeat module with the ankyrin repeat sequence motif of SEQ ID NO:3,wherein said repeat module is preceded by a repeat module with theankyrin repeat sequence motif of SEQ ID NO:2 and/or followed by a repeatmodule with the ankyrin repeat sequence motif of SEQ ID NO:4.

Further preferred is a recombinant binding protein, wherein said ankyrinrepeat domain or said designed ankyrin repeat domain comprises a repeatmodule with the ankyrin repeat sequence motif

(SEQ ID NO: 5) 1D23G4TPLHLAA56GH7EIVEVLLK8GADVNA wherein

1 represents an amino acid residue selected from the group consisting ofA, N, R, V, Y, E, H, I, K, L, Q, S and T; preferably A, N, R, V and Y;more preferably A and R;

2 represents an amino acid residue selected from the group consisting ofS, A, N, R, D, F, L, P, T and Y; preferably S, A, N and R;

3 represents an amino acid residue selected from the group consisting ofT, V, S, A, L and F; preferably T, V, S, A and L; more preferably T, Vand S;

4 represents an amino acid residue selected from the group consisting ofW, F and H;

5 represents an amino acid residue selected from the group consisting ofP, I, A, L, S, T, V and Y; preferably P and I;

6 represents an amino acid residue selected from the group consisting ofW, F, I, L, T and V;

7 represents the amino acid residue L or P; and

8 represents an amino acid residue selected from the group consisting ofA, H, N and Y.

Further preferred is a recombinant binding protein, wherein said ankyrinrepeat domain or said designed ankyrin repeat domain comprises a repeatmodule with the ankyrin repeat sequence motif

(SEQ ID NO: 6) 1D23G4TPLHLAA56GHLEIVEVLLK7GADVNA wherein

1 represents an amino acid residue selected from the group consisting ofH, Q, A, K, R, D, I, L, M, N, V and Y; preferably H, Q, A, K and R; morepreferably A and R;

2 represents an amino acid residue selected from the group consisting ofY, F and H;

3 represents an amino acid residue selected from the group consisting ofQ, F and T;

4 represents an amino acid residue selected from the group consisting ofW, M, G, H, N and T; preferably W and M;

5 represents an amino acid residue selected from the group consisting ofT, A, M, L and V; preferably T, A and M;

6 represents an amino acid residue selected from the group consisting ofI, L, V, D and T; preferably I, L and V; and

7 represents an amino acid residue selected from the group consisting ofA, H, N and Y.

Even more preferred is a recombinant binding protein, wherein saidankyrin repeat domain or said designed ankyrin repeat domain comprises arepeat module with the ankyrin repeat sequence motif of SEQ ID NO:6,wherein said repeat module is preceded by a repeat module with theankyrin repeat sequence motif of SEQ ID NO:5.

Further preferred is a recombinant binding protein, wherein said ankyrinrepeat domain or said designed ankyrin repeat domain comprises a repeatmodule with the ankyrin repeat sequence motif

(SEQ ID NO: 7) 1D23GWTPLHLAA45GHLEIVEVLLK6GADVNA wherein

1 represents an amino acid residue selected from the group consisting ofK, M, N, R and V;

2 represents an amino acid residue selected from the group consisting ofY, H, M and V;

3 represents an amino acid residue selected from the group consisting ofF, L, M and V;

4 represents an amino acid residue selected from the group consisting ofR, H, V, A, K and N; preferably R, H, V and A;

5 represents an amino acid residue selected from the group consisting ofF, D, H, T, Y, M and K; preferably F, D, H, T and Y; and

6 represents an amino acid residue selected from the group consisting ofA, H, N and Y.

Further preferred is a recombinant binding protein, wherein said ankyrinrepeat domain or said designed ankyrin repeat domain comprises a repeatmodule with the ankyrin repeat sequence motif

(SEQ ID NO: 8) 1D23G4TPLHLAA56GHLEIVEVLLK7GADVN8 wherein

1 represents an amino acid residue selected from the group consisting ofT, A, H, I, N and S;

2 represents an amino acid residue selected from the group consisting ofF, I, Q, R, V and N;

3 represents an amino acid residue selected from the group consisting ofA, G, N, Q and V;

4 represents the amino acid residue W or Y;

5 represents an amino acid residue selected from the group consisting ofA, S, T and M;

6 represents an amino acid residue selected from the group consisting ofN, V, S, F, M and W;

7 represents an amino acid residue selected from the group consisting ofA, H, N and Y; and

8 represents the amino acid residue T or A.

Further preferred is a recombinant binding protein, wherein said ankyrinrepeat domain or said designed ankyrin repeat domain comprises a repeatmodule with the ankyrin repeat sequence motif

(SEQ ID NO: 9) 1D23G4TPLHL5A67GHLEIVEVLLK8GADVNA wherein

1 represents an amino acid residue selected from the group consisting ofK, A, V and N;

2 represents an amino acid residue selected from the group consisting ofN, I and Y;

3 represents an amino acid residue selected from the group consisting ofT, F, Y and W;

4 represents an amino acid residue selected from the group consisting ofW, D and Y;

5 represents the amino acid residue S or A;

6 represents an amino acid residue selected from the group consisting ofD, I and M;

7 represents an amino acid residue selected from the group consisting ofL, T and Y; and

8 represents an amino acid residue selected from the group consisting ofA, H, Y and N;

Even more preferred is a recombinant binding protein, wherein saidankyrin repeat domain or said designed ankyrin repeat domain comprises arepeat module with the ankyrin repeat sequence motif of SEQ ID NO:8,wherein said repeat module is preceded by a repeat module with theankyrin repeat sequence motif of SEQ ID NO:7 and/or followed by a repeatmodule with the ankyrin repeat sequence motif of SEQ ID NO:9.

Further preferred is a recombinant binding protein, wherein said ankyrinrepeat domain or said designed ankyrin repeat domain comprises a repeatmodule with the ankyrin repeat sequence motif

(SEQ ID NO: 10) 1DFK2DTPLHLAA34GH5EIVEVLLK6GADVNA wherein

1 represents an amino acid residue selected from the group consisting ofL, S and T;

2 represents an amino acid residue selected from the group consisting ofG, S and C; preferably G and S;

3 represents the amino acid residue S or A;

4 represents an amino acid residue selected from the group consisting ofQ, S, M and N; preferably Q and S;

5 represents an amino acid residue selected from the group consisting ofL, M and Q; and

6 represents an amino acid residue selected from the group consisting ofA, H, N, Y and D.

Further preferred is a recombinant binding protein, wherein said ankyrinrepeat domain or said designed ankyrin repeat domain comprises a repeatmodule with the ankyrin repeat sequence motif

(SEQ ID NO: 11) 1D2L34TPLHLA567GHLEIVEVLLK8GADVNA wherein

1 represents an amino acid residue selected from the group consisting ofY, H, F, I, L and W; preferably Y and H;

2 represents an amino acid residue selected from the group consisting ofM, D, I, L, V; preferably M and D;

3 represents an amino acid residue selected from the group consisting ofG, S and V;

4 represents the amino acid residue W or F;

5 represents an amino acid residue selected from the group consisting ofA, G and T;

6 represents the amino acid residue D or W;

7 represents the amino acid residue L or F; and

8 represents an amino acid residue selected from the group consisting ofA, H, N and Y.

Even more preferred is a recombinant binding protein, wherein saidankyrin repeat domain or said designed ankyrin repeat domain comprises arepeat module with the ankyrin repeat sequence motif of SEQ ID NO:11,wherein said repeat module is preceded by a repeat module with theankyrin repeat sequence motif of SEQ ID NO:10.

Further preferred is a recombinant binding protein, wherein said ankyrinrepeat domain or said designed ankyrin repeat domain comprises a repeatmodule with the ankyrin repeat sequence motif

(SEQ ID NO: 12) 1D23G4TPL5LAA67GHLEIVEVLLK8GADVNA wherein

1 represents an amino acid residue selected from the group consisting ofK, S, I, N, T and V; preferably K and S;

2 represents an amino acid residue selected from the group consisting ofK, N, W, A, H, M, Q and S; preferably K and N;

3 represents an amino acid residue selected from the group consisting ofF, Q, L, H and V; preferably F, Q and L;

4 represents the amino acid residue F or T;

5 represents the amino acid residue Q or H;

6 represents the amino acid residue Y or S;

7 represents an amino acid residue selected from the group consisting ofN, H, Y and M; preferably N and H; and

8 represents an amino acid residue selected from the group consisting ofA, H, N and Y.

Further preferred is a recombinant binding protein, wherein said ankyrinrepeat domain or said designed ankyrin repeat domain comprises a repeatmodule with the ankyrin repeat sequence motif

(SEQ ID NO: 13) 1D23GWT4LHLAADLG5LEIVEVLLK6GADVNA wherein

1 represents an amino acid residue selected from the group consisting ofF, Y, H and W; preferably F, Y and H;

2 represents an amino acid residue selected from the group consisting ofI, M, D and V; preferably I, M and D;

3 represents the amino acid residue F or L;

4 represents the amino acid residue L or P;

5 represents an amino acid residue selected from the group consisting ofH, L and Y; and

6 represents an amino acid residue selected from the group consisting ofA, H, N, C and Y.

Even more preferred is a recombinant binding protein, wherein saidankyrin repeat domain or said designed ankyrin repeat domain comprises arepeat module with the ankyrin repeat sequence motif of SEQ ID NO:13,wherein said repeat module is preceded by a repeat module with theankyrin repeat sequence motif of SEQ ID NO:12.

Another preferred embodiment is a recombinant binding protein comprisingat least one repeat domain with binding specificity for VEGF-Axxx,wherein said repeat domain competes for binding to VEGF-Axxx with arepeat domain selected from the group consisting of SEQ ID NOs:16, 22,23, 29, 30 and 33, or a repeat domain selected from the group consistingof SEQ ID NOs:16, 22, 23, 29, 30, 33, 34, 36, 39 and 40.

The term “compete for binding” means the inability of two differentbinding domains of the invention to bind simultaneously to the sametarget, while both are able to bind the same target individually. Thus,such two binding domains compete for binding to said target. Methods,such as competition ELISA or competition SPR measurements (e.g. by usingthe Proteon instrument from BioRad), to determine if two binding domainscompete for binding to a target are well known to the practitioner inthe art.

A recombinant binding protein that competes for binding to VEGF-Axxxwith a selected repeat protein can be identified by methods well know tothe person skilled in the art, such as a competition Enzyme-LinkedImmunoSorbent Assay (ELISA).

Another preferred embodiment is a recombinant binding protein comprisinga repeat domain with binding specificity for VEGF-Axxx selected from thegroup consisting of SEQ ID NOs:14 to 33, or selected from the groupconsisting of SEQ ID NOs:14 to 40.

Further preferred is a recombinant binding protein, wherein said repeatdomain with binding specificity for VEGF-Axxx comprises an amino acidsequence that has at least 70% amino acid sequence identity with arepeat domain of said group of repeat domains. Preferably, said aminoacid sequence identity is at least 75%, more preferably 80%, morepreferably 85%, more preferably 90%, and most preferably 95%.

Further preferred is a recombinant binding protein, wherein said repeatdomain with binding specificity for VEGF-Axxx comprises a repeat modulethat has at least 70% amino acid sequence identity with a repeat moduleof a repeat domain of said group of repeat domains. Preferably, saidamino acid sequence identity is at least 75%, more preferably 80%, morepreferably 85%, more preferably 90%, and most preferably 95%.

In a further preferred embodiment of a recombinant binding proteincomprising a repeat domain according to the present invention, one ormore of the amino acid residues of the repeat modules of said repeatdomain are exchanged by an amino acid residue found at the correspondingposition on alignment of a repeat unit. Preferably, up to 30% of theamino acid residues are exchanged, more preferably, up to 20%, and evenmore preferably, up to 10% of the amino acid residues are exchanged.Most preferably, such a repeat unit is a naturally occurring repeatunit. Even more preferably, said repeat domain has binding specificityfor VEGF-Axxx or VEGFR-2.

In still another particular embodiment, up to 30% of the amino acidresidues, more preferably, up to 20%, and even more preferably, up to10% of the amino acid residues are exchanged with amino acids which arenot found in the corresponding positions of repeat units.

In further embodiments, any of the VEGF-Axxx binding proteins or domainsdescribed herein may be covalently bound to one or more additionalmoieties, including, for example, a moiety that also binds to VEGFR-2(e.g. a VEGFR-2 binding polypeptide), a moiety that binds to a differenttarget, such as PIGF, human serum albumin, a cellular receptor (e.g.Her2), an immunoglobulin (e.g. IgG1), a cytokine (e.g. TNF-alpha or aninterleukin) or a growth factor to create a dual-specificity bindingagent, a labeling moiety (e.g. a fluorescent label such as fluorescein,or a radioactive tracer), a moiety that facilitates protein purification(e.g. a small peptide tag, such as a His- or strep-tag), a moiety thatprovides effector functions for improved therapeutic efficacy (e.g. theFc part of an antibody to provide antibody-dependent cell-mediatedcytotoxicity, a toxic protein moiety such as Pseudomonas aeruginosaexotoxin A (ETA) or a small molecular toxic agent such as maytansinoidsor DNA alkylating agents) or a moiety that provides improvedpharmacokinetics. Improved pharmacokinetics may be assessed according tothe perceived therapeutic need. Often it is desirable to increasebioavailability and/or increase the time between doses, possibly byincreasing the time that a protein remains available in the serum afterdosing. In some instances, it is desirable to improve the continuity ofthe serum concentration of the protein over time (e.g., decrease thedifference in serum concentration of the protein shortly afteradministration and shortly before the next administration). Moietiesthat tend to slow clearance of a protein from the blood includehydroxyethyl starch (HES), polyethylene glycol (PEG), sugars (e.g.sialic acid), well-tolerated protein moieties (e.g. Fc fragment or serumalbumin), and binding domains or peptides with specificity and affinityfor abundant serum proteins, such as antibody Fc fragments or serumalbumin. The recombinant binding protein of the invention may beattached to a moiety that reduces the clearance rate of polypeptides ina mammal (e.g. in mouse, rat, or human) by greater than three-foldrelative to the unmodified polypeptides.

One ore more polyethylene glycol moieties may be attached at differentpositions in the binding protein, and such attachment may be achieved byreaction with amines, thiols or other suitable reactive groups.Attachment of polyethylene glycol moieties (PEGylation) may besite-directed, wherein a suitable reactive group is introduced into theprotein to create a site where PEGylation preferentially occurs, or isoriginally present in the binding protein. The thiol group may bepresent in a cysteine residue; and the amine group may be, for example,a primary amine found at the N-terminus of the polypeptide or an aminegroup present in the side chain of an amino acid, such as lysine orarginine. In a preferred embodiment, the binding protein is modified soas to have a cysteine residue at a desired position, permitting sitedirected PEGylation on the cysteine, for example by reaction with apolyethylene glycol derivative carrying a maleimide function. Thepolyethylene glycol moiety may vary widely in molecular weight (i.e.from about 1 kDa to about 100 kDa) and may be branched or linear.Preferably, the polyethylene glycol has a molecular weight of about 1 toabout 50 kDa, preferably about 10 to about 40 kDa, even more preferablyabout 15 to about 30 kDa, and most preferably about 20 kDa.

In a further embodiment, the invention relates to nucleic acid moleculesencoding the particular recombinant binding proteins. Further, a vectorcomprising said nucleic acid molecule is considered.

Further, a pharmaceutical composition comprising one or more of theabove mentioned binding proteins, in particular recombinant bindingproteins comprising repeat domains, or nucleic acid molecules encodingthe particular recombinant binding proteins, and optionally apharmaceutical acceptable carrier and/or diluent is considered.

Pharmaceutical acceptable carriers and/or diluents are known to theperson skilled in the art and are explained in more detail below. Evenfurther, a diagnostic composition comprising one or more of the abovementioned recombinant binding proteins, in particular binding proteinscomprising repeat domains, is considered.

The binding protein of the invention suppresses or prevents VEGF inducedpathological angiogenesis, vascular leakage (edema), pulmonaryhypertension, tumor formation and/or inflammatory disorders. With“suppression” it is understood that the recombinant protein prevents thementioned pathologies to some extent, e.g. to 10% or 20%, morepreferably 50%, in particular 70%, 80% or 90%, or even 95%.

The term “edema” means a condition that is caused by vascular leakage.Vasodilation and increased permeability during inflammation can bepredominant pathogenetic mechanisms. For instance, edema contributes toinfarct expansion after stroke and may cause life-threateningintracranial hypertension in cancer patients. Further, extravasation ofplasma proteins favors metastatic spread of occult tumors, and airwaycongestion may cause fatal asthmatic attacks. The increased vascularleakage which occurs during inflammation can lead to respiratorydistress, ascites, peritoneal sclerosis (in dialysis patients), adhesionformation (abdominal surgery) and metastatic spreading.

The term “angiogenesis” means a fundamental process by which new bloodvessels are formed. The primary angiogenic period in humans takes placeduring the first three months of embryonic development but angiogenesisalso occurs as a normal physiological process during periods of tissuegrowth, such as an increase in muscle or fat and during the menstrualcycle and pregnancy.

The term “pathological angiogenesis” refers to the formation and growthof blood vessels during the maintenance and the progression of severaldisease states. Particular examples of pathological angiogenesis arefound in blood vessels (atherosclerosis, hemangioma,hemangioendothelioma), bone and joints (rheumatoid arthritis, synovitis,bone and cartilage destruction, osteomyelitis, pannus growth, osteophyteformation, neoplasms and metastasis), skin (warts, pyogenic granulomas,hair growth, Kaposi's sarcoma, scar keloids, allergic edema, neoplasms),liver, kidney, lung, ear and other epithelia (inflammatory andinfectious processes including hepatitis, glomerulonephritis, pneumonia;and asthma, nasal polyps, otitis, transplantation disorders, liverregeneration disorders, neoplasms and metastasis), uterus, ovary andplacenta (dysfunctional uterine bleeding due to intra-uterinecontraceptive devices, follicular cyst formation, ovarianhyperstimulation syndrome, endometriosis, neoplasms), brain, nerves andeye (retinopathy of prematurity, diabetic retinopathy, choroidal andother intraocular disorders, leukomalacia, neoplasms and metastasis),heart and skeletal muscle due to work overload, adipose tissue(obesity), endocrine organs (thyroiditis, thyroid enlargement, pancreastransplantation disorders), hematopoiesis (Kaposi syndrome in AIDS),hematologic malignancies (leukemias), and lymph vessels (tumormetastasis, lymphoproliferative disorders).

The term “retinal ischemic diseases” means that the retina's supply ofblood and oxygen is decreased, the peripheral portions of the retinalose their source of nutrition and stop functioning properly. Aparticular example of a retinal ischemic disease is retinopathy. Commondiseases which lead to retinopathy are diabetic retinopathy, centralretinal vein occlusion, stenosis of the carotid artery, and sickle cellretinopathy. Diabetic retinopathy is a major cause of visual loss indiabetic patients. In the ischemic retina the growth of new bloodvessels occurs (neovascularisation). These vessels often grow on thesurface of the retina, at the optic nerve, or in the front of the eye onthe iris. The new vessels cannot replace the flow of necessary nutrientsand, instead, can cause many problems such as vitreous hemorrhage,retinal detachment, and uncontrolled glaucoma. These problems occurbecause new vessels are fragile and are prone to bleed. If caught in itsearly stages, proliferative diabetic retinopathy can sometimes bearrested with panretinal photocoagulation. However, in some cases,vitrectomy surgery is the only option.

Beside these retinopathies, vascular diseases of the eye also includeocular neovascularization diseases, such as macular degeneration anddiabetic macular edema (DME). Macular degeneration results from theneovascular growth of the choroid vessel underneath the macula. Thereare two types of macular degeneration: dry and wet. While wet maculardegeneration only comprises 15% of all macular degeneration, nearly allwet macular degeneration leads to blindness. In addition, wet maculardegeneration nearly always results from dry macular degeneration. Onceone eye is affected by wet macular degeneration, the condition almostalways affects the other eye. Wet macular degeneration is often calledage-related wet macular degeneration of wet-AMD as it is mostly found inelderly persons.

Diabetic retinopathy (DR) and DME are leading causes of blindness in theworking-age population of most developed countries. The increasingnumber of individuals with diabetes worldwide suggests that DR and DMEwill continue to be major contributors to vision loss and associatedfunctional impairment for years to come. Several biochemical mechanisms,including protein kinase C-β activation, increased vascular endothelialgrowth factor production, oxidative stress, and accumulation ofintracellular sorbitol and advanced glycosylation end products, maycontribute to the vascular disruptions that characterize DR/DME. Theinhibition of these pathways holds the promise of intervention for DRand DME.

The term “pulmonary hypertension” means a disorder in which the bloodpressure in the pulmonary arteries is abnormally high. In the absence ofother diseases of the heart or lungs it is called primary pulmonaryhypertension. Diffuse narrowing of the pulmonary arterioles occurs as aresult of pathological arteriogenesis followed by pulmonary hypertensionas a response to the increased resistance to blood flow. The incidenceis 8 out of 100′000 people. However, pulmonary hypertension can alsooccur as a complication of Chronic Obstructive Pulmonary Diseases (COPD)such as emphysema, chronic bronchitis or diffuse interstitial fibrosisand in patients with asthmatiform COPD. The incidence of COPD isapproximately 5 out of 10′000 people.

Furthermore the binding proteins of the invention can be used to treatinflammation and more specifically inflammatory disorders.

The term “inflammation” as used herein means, the local reaction toinjury of living tissues, especially the local reaction of the smallblood vessels, their contents, and their associated structures. Thepassage of blood constituents through the vessel walls into the tissuesis the hallmark of inflammation, and the tissue collection so formed istermed the exudates or edema. Any noxious process that damages livingtissue, e.g. infection with bacteria, excessive heat, cold, mechanicalinjury such as crushing, acids, alkalis, irradiation, or infection withviruses can cause inflammation irrespective of the organ or tissueinvolved. It should be clear that diseases classified as “inflammatorydiseases” and tissue reactions ranging from burns to pneumonia, leprosy,tuberculosis, and rheumatoid arthritis are all “inflammations”.

The binding proteins according to the invention can be used to treattumor formation. The term “tumor” means a mass of abnormal tissue thatarises without obvious cause from pre-existing body cells, has nopurposeful function, and is characterized by a tendency to autonomousand unrestrained growth. Tumors are quite different from inflammatory orother swellings because the cells in tumors are abnormal in theirappearance and other characteristics. Abnormal cells, i.e. the kind ofcells that generally make up tumors, differ from normal cells in havingundergone one or more of the following alterations: (1) hypertrophy, oran increase in the size of individual cells; (2) hyperplasia or anincrease in the number of cells within a given zone; (3) anaplasia, or aregression of the physical characteristics of a cell toward a moreprimitive or undifferentiated type. Tumors may be benign, for examplelipomas, angiomas, osteomas, chondromas, and adenomas. Examples ofmalignant tumors are carcinomas (such as the breast tumors, carcinomasin the respiratory and gastrointestinal tracts, the endocrine glands,and the genitourinary system), sarcomas (in connective tissues,including fibrous tissues, adipose (fat) tissues, muscle, blood vessels,bone, and cartilage), carcinosarcoma (in both epithelial and connectivetissue) leukemias and lymphomas, tumors of nerve tissues (including thebrain), and melanoma (a cancer of the pigmented skin cells). The use ofthe binding proteins of the present invention against tumors can also bein combination with any other tumor therapy known in the art such asirradiation, photo-dynamic therapy, chemotherapy or surgery.

A pharmaceutical composition comprises binding proteins as describedabove and a pharmaceutically acceptable carrier, excipient or stabilizer(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]).Suitable carriers, excipients or stabilizers known to the skilled manare saline, Ringer's solution, dextrose solution, Hank's solution, fixedoils, ethyl oleate, 5% dextrose in saline, substances that enhanceisotonicity and chemical stability, buffers and preservatives. Othersuitable carriers include any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids and amino acid copolymers. Apharmaceutical composition may also be a combination formulation,comprising an additional active agent, such as an anti-cancer agent oran anti-angiogenic agent (for example human VEGF-Axxxb; preferably,human VEGF-A165b).

A preferred pharmaceutical composition for the treatment of eye diseasescomprises binding proteins as described above and a detergent such aspolysorbate 20 (e.g. about 0.04%), a buffer such as histidine, phosphateor lactic acid and a sugar such as sucrose or trehalose. Preferably,such a composition comprises binding proteins as described above andPBS. Said pharmaceutical compositions may be administered locally,either topically to a portion of the eye or be injected into the eye forinstance into the subconjunctivital, peri- or retrobulbar space ordirectly into the eye. Alternatively, said compositions may beadministered systemically by parental administration. Preferably, saidpharmaceutical composition is applied to the eye by an intravitreousinjection. Also preferably, said pharmaceutical composition is appliedto the eye topically and as an eye drop. The eye drop may be applied tothe cornea (clear part in the centre of the eye) thereby allowing themolecules to permeate into the eye. For the treatment of a diseaseaffecting the posterior of the eye, it may be most desirable that thebinding protein penetrates the sclera when injected under theconjunctiva or around the globe. The administering of the bindingprotein may be performed after a preliminary step of modulating thesurface of the eye to improve penetration of the molecules. Preferably,the epithelial layer such as the corneal epithelium is modulated by apenetration enhancer to allow for a sufficient and rapid penetration ofthe molecules as for example described above. The use of the bindingproteins of the present invention against eye diseases can also be incombination with any other therapy known in the art such asphoto-dynamic therapy.

The formulations to be used for in vivo administration must be asepticor sterile. This is readily accomplished by filtration through sterilefiltration membranes.

Sustained-release preparations may be prepared. In one embodiment of theinvention, an intraocular implant can be used for providing the bindingprotein of the invention. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing a polypeptide of the invention, which matrices arein the form of shaped articles, e.g. films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as the LUPRON DEPOT® (injectable microspherescomposed of lactic acid-glycolic acid copolymer and leuprolide acetate),and poly-D-(-)-3-hydroxybutyric acid.

The pharmaceutical composition may be administered by any suitablemethod within the knowledge of the skilled man. The preferred route ofadministration is parenterally. In parental administration, themedicament of this invention will be formulated in a unit dosageinjectable form such as a solution, suspension or emulsion, inassociation with the pharmaceutically acceptable excipients as definedabove. The dosage and mode of administration will depend on theindividual to be treated and the particular disease. Generally, thepharmaceutical composition is administered so that the binding proteinof the present invention is given at a dose between 1 μg/kg and 20mg/kg, more preferably between 10 μg/kg and 5 mg/kg, most preferablybetween 0.1 and 2 mg/kg. Preferably, it is given as a bolus dose.Continuous infusion may also be used and includes continuoussubcutaneous delivery via an osmotic minipump. If so, the pharmaceuticalcomposition may be infused at a dose between 5 and 20 μg/kg/minute, morepreferably between 7 and 15 μg/kg/minute. In particular, thepharmaceutical composition is administered by injections into the eye sothat the binding protein of the invention is given at a dose between 0.1mg and 10 mg per injection, more preferably between 0.3 and 6 mg perinjection, most preferably between 1 mg and 4 mg per injection. Further,the pharmaceutical composition is administered by eye drops to the eyeso that a single drop of a solution containing a concentration of thebinding protein of the invention between 10 and 120 mg/ml, morepreferably between 20 and 100 mg/ml, most preferably between 40 and 80mg/ml is applied to the eye.

In another embodiment of the invention a binding protein inhibiting theactivity of VEGF-Axxx, as described above, can be used in combinationwith a binding protein or small molecule inhibiting the activity ofPIGF, with the same inhibition levels of PIGF as described above forVEGF-Axxx. This embodiment is based on the fact that PIGF is found to beangiogenic at sites where VEGF-Axxx levels are increased. Further, abinding protein inhibiting the activity of VEGF-Axxx, as describedabove, can be used in combination with a binding protein or smallmolecule inhibiting the activity of platelet-derived growth factor(PDGF), VEGF-C or other members of the VEGF family of proteins, tumornecrosis factor alpha (TNFalpha), delta-ligand like 4 (Dll4),interleukin 6 (IL-6), neuropilin or angiopoietin 2 (Ang2).

The invention further provides novel methods of treatment. In oneaspect, a method of treating a retinopathy is provided, the methodcomprising administering, to a patient in need thereof, atherapeutically effective amount of a binding protein of the invention,in particular a binding protein that inhibits the interaction betweenhuman VEGF-Axxx and human VEGFR-2, but not the interaction between humanVEGF-Axxxb and human VEGFR-2, and the binding protein inhibits VEGFR-2mediated angiogenesis.

The invention further relates to methods for using a binding protein asdescribed to inhibit a VEGF-A biological activity in a cell or toinhibit a biological activity mediated by VEGFR-2. The cell may besituated in vivo or ex vivo, and may be, for example, a cell of a livingorganism, a cultured cell or a cell in a tissue sample. The method maycomprise contacting said cell with any of the VEGF-A/VEGFR-2 interactioninhibiting binding proteins disclosed herein, in an amount and for atime sufficient to inhibit such biological activity.

The invention provides a method for treating a subject having acondition which responds to the inhibition of VEGF-Axxx or VEGFR-2. Sucha method comprises administering to said subject an effective amount ofa binding protein described herein. A condition may be one that ischaracterized by inappropriate angiogenesis. A condition may be ahyper-proliferative condition. Examples of conditions (or disorders)suitable for treatment include autoimmune disorders, inflammatorydisorders, retinopathies (particularly proliferative retinopathies), andcancers, in particular one of the diseases described above. Any of thebinding proteins described herein may be used for the preparation of amedicament for the treatment of such a disorder, particularly a disorderselected from the group consisting of: an autoimmune disorder, aninflammatory disorder, a retinopathy, and a cancer. Preferred conditions(or disorders) suitable for treatment are first-line metastatic renalcell carcinoma, relapsed glioblastoma multiforme, adjuvant colon cancer,adjuvant HER2-negative breast cancer, adjuvant HER2-positive breastcancer, adjuvant non-small cell lung cancer, diffuse large B-celllymphoma, first-line advanced gastric cancer, first-line HER2-negativemetastatic breast cancer, first-line HER2-positive metastatic breastcancer, first-line metastatic ovarian cancer, gastrointestinal stromaltumors, high risk carcinoid, hormone refractory prostate cancer, newlydiagnosed glioblastoma multiforme, metastatic head and neck cancer,relapsed platinum-sensitive ovarian cancer, second-line metastaticbreast cancer, extensive small cell lung cancer, non-squamous, non-smallcell lung cancer with previously treated CNS metastases and relapsedmultiple myeloma, prostate cancer, non-small cell lung cancer (NSCLC),colorectal cancer and pancreatic cancer, advanced ovarian cancer (AOC),AOC patients with symptomatic malignant ascites and non-Hodgkin'slymphoma.

The recombinant binding protein according to the invention may beobtained and/or further evolved by several methods such as display onthe surface of bacteriophages (WO 90/02809, WO 07/006665) or bacterialcells (WO 93/10214), ribosomal display (WO 98/48008), display onplasmids (WO 93/08278) or by using covalent RNA-repeat protein hybridconstructs (WO 00/32823), or intracellular expression andselection/screening such as by protein complementation assay (WO98/341120). Such methods are known to the person skilled in the art.

A library of ankyrin repeat proteins used for the selection/screening ofa recombinant binding protein according to the invention may be obtainedaccording to protocols known to the person skilled in the art (WO02/020565, Binz, H. K. et al., JMB, 332, 489-503, 2003, and Binz et al.,2004, loc. cit). The use of such a library for the selection VEGF-Axxxspecific DARPins is given in Example 1. In analogy, the ankyrin repeatsequence motifs as presented above can used to build libraries ofankyrin repeat proteins that may be used for the selection or screeningof VEGF-Axxx specific DARPins. Furthermore, repeat domains of thepresent invention may be modularly assembled from repeat modulesaccording the current inventions and appropriate capping modules(Forrer, P., et al., FEBS letters 539, 2-6, 2003) using standardrecombinant DNA technologies (e.g. WO 02/020565, Binz et al., 2003, loc.cit. and Binz et al., 2004, loc. cit).

The invention is not restricted to the particular embodiments describedin the Examples. Other sources may be used and processed following thegeneral outline described below.

EXAMPLES

All of the starting materials and reagents disclosed below are known tothose skilled in the art, and are available commercially or can beprepared using well-known techniques.

Materials

Chemicals were purchased from Fluka (Switzerland). Oligonucleotides werefrom Microsynth (Switzerland). Unless stated otherwise, DNA polymerases,restriction enzymes and buffers were from New England Biolabs (USA) orFermentas (Lithuania). The cloning and protein production strain was E.coli XL1-blue (Stratagene, USA). VEGF variants were from R&D Systems(Minneapolis, USA) or were produced in Chinese Hamster Ovary Cells or inPichia pastoris and purified according to standard protocols (Rennel, E.S. et al., European J. Cancer 44, 1883-94, 2008; Pichia expressionsystem from Invitrogen). Biotinylated VEGF variants were obtainedchemically via coupling of the biotin moiety to primary amines of thepurified VEGF variants using standard biotinylation reagents and methods(Pierce, USA).

Molecular Biology

Unless stated otherwise, methods are performed according to describedprotocols (Sambrook J., Fritsch E. F. and Maniatis T., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, NewYork).

Designed Ankyrin Repeat Protein Libraries

The N2C and N3C designed ankyrin repeat protein libraries are described(WO 02/20565; Binz et al. 2003, loc. cit.; Binz et al. 2004, loc. cit.).The digit in N2C and N3C describes the number of randomized repeatmodules present between the N-terminal and C-terminal capping modules.The nomenclature used to define the positions inside the repeat unitsand modules is based on Binz et al. 2004, loc. cit. with themodification that borders of the repeat modules and repeat units areshifted by one amino acid position. For example, position 1 of a repeatmodule of Binz et al. 2004 (loc. cit.) corresponds to position 2 of arepeat module of the current disclosure and consequently position 33 ofa repeat module of Binz et al. 2004, loc. cit. corresponds to position 1of a following repeat module of the current disclosure.

All the DNA sequences were confirmed by sequencing, and the calculatedmolecular weight of all described proteins was confirmed by massspectrometry.

Example 1: Selection of Binding Proteins Comprising a Repeat Domain withBinding Specificity for VEGF-Axxx

Using ribosome display (Hanes, J. and Plückthun, A., PNAS 94, 4937-42,1997) many designed ankyrin repeat proteins (DARPins) with bindingspecificity for VEGF-Axxx were selected from the N2C or N3C DARPinlibraries described by Binz et al. 2004 (loc. cit.). The binding of theselected clones toward specific (VEGF-Axxx) and unspecific (MBP, E. colimaltose binding protein) targets was assessed by crude extract ELISAindicating that VEGF-Axxx binding proteins were successfully selected(FIG. 1). SEQ ID NO:14 to 40 constitute amino acid sequences of selectedbinding proteins comprising a repeat domain with binding specificity forVEGF-Axxx. Sequence analysis of selected binders revealed specificankyrin repeat sequence motifs inherent to certain selected families ofbinders. Such ankyrin repeat sequence motifs present in repeat domainswith binding specificity for VEGF-Axxx are provided in SEQ ID NO:1 to13.

Selection of VEGF-Axxx Specific Ankyrin Repeat Proteins by RibosomeDisplay

The selection of VEGF-Axxx specific ankyrin repeat proteins wasperformed by ribosome display (Hanes and Plückthun, loc. cit.) using dogVEGF-A164 or human VEGF-A165 as target proteins, the library of designedankyrin repeat proteins as described (WO 02/020565, Binz et al., 2003,loc. cit. and Binz et al., 2004, loc. cit) and established protocols(Zahnd, C., Amstutz, P. and Plückthun, A., Nat. Methods 4, 69-79, 2007).Ribosome-display selection rounds were performed on dog or human VEGFvariants (including biotinylated variants immobilized over neutravidinor streptavidin) with both the N2C and N3C DARPin libraries usingestablished protocols (Binz et al. 2004, loc. cit.). The number ofreverse transcription (RT)-PCR cycles after each selection round wasconstantly reduced from 40 to 30, adjusting to the yield due toenrichment of binders. Four initial selection rounds on dog VEGF yieldedpools of nanomolar-affinity DARPins, as revealed by ELISA and SPRmeasurements of single clones. To find DARPins with further improvedaffinities, additional off-rate selections were performed onbiotinylated human or dog VEGF immobilized over neutravidin orstreptavidin, taking pools after the second and third initialribosome-display selection rounds, followed by an on-rate selectionround on human VEGF.

Selected clones bind specifically to VEGF-Axxx as shown by crude extractELISA Individual selected DARPins specifically binding VEGF-Axxx wereidentified by an enzyme-linked immunosorbent assay (ELISA) using crudeEscherichia coli extracts of DARPin expression cells using standardprotocols. Selected clones were cloned into the pQE30 (Qiagen)expression vector, transformed into E. coli XL1-Blue (Stratagene) andthen grown overnight at 37° C. in a 96-deep-well plate (each clone in asingle well) containing 1 ml growth medium (2YT containing 1% glucoseand 100 μg/ml ampicillin). 1 ml of fresh 2YT containing 50 μg/mlampicillin was inoculated with 100 μl of the overnight culture in afresh 96-deep-well plate. After incubation for 2 h at 37° C., expressionwas induced with IPTG (1 mM final concentration) and continued for 3 h.Cells were harvested, resuspended in 100 μl B-PERII (Pierce) andincubated for 15 min at room temperature with shaking. Then, 900 μlPBS-TB (PBS supplemented with 0.2% BSA, 0.1% Tween 20, pH 7.4) wereadded and cell debris were removed by centrifugation. 100 μl of eachlysed clone were applied to a well of a NeutrAvidin coated MaxiSorpplate containing either a VEGF-Axxx variant or the unrelated MBPimmobilized via their biotin moiety and incubated for 1 h at RT. Afterextensive washing with PBS-T (PBS supplemented with 0.1% Tween 20, pH7.4) the plate was developed using standard ELISA procedures using themonoclonal anti-RGS(His)₄ antibody (34650, Qiagen) as primary antibodyand a polyclonal goat anti-mouse antibody conjugated with alkalinephosphatase (A3562, Sigma) as secondary reagent. Binding was thendetected by using disodium 4-nitrophenyl phosphate (4NPP, Fluka) as asubstrate for alkaline phosphatase. The color development was measuredat 405 nm. The results from an example crude extract ELISA used toidentify DARPins binding to VEGF-Axxx is shown in FIG. 1.

Screening of several hundred clones by such a crude cell extract ELISArevealed more than hundred different DARPins with specificity forVEGF-Axxx. These binding proteins were chosen for further analysis.Examples of amino acid sequences of selected ankyrin repeat domains thatspecifically bind to VEGF-Axxx are provided in SEQ ID NO:14 to 40.

Deducing Repeat Sequence Motives from Selected Repeat Domains withBinding Specificity for VEGF-Axxx

The amino acid sequences of selected repeat domains with bindingspecificity for VEGF-Axxx were further analyzed by sequence analyzingtools known to the practitioner in the art (WO 02/020565; Forrer et al.,2003, loc. cit.; Forrer, P., Binz, H. K., Stumpp, M. T. and Plückthun,A., ChemBioChem, 5(2), 183-189, 2004). Nevertheless, in contrast to WO02/020565 where naturally occurring repeat motifs were used to deducerepeat sequence motifs, here the repeat sequence motifs were deducedfrom the repeat units of selected repeat domains with bindingspecificity for VEGF-Axxx. Thereby families of selected repeat domainscomprising a common repeat sequence motif were determined. Such repeatsequence motifs present in repeat domains with binding specificity forVEGF-Axxx are provided in SEQ ID NO:1 to 13.

High Level and Soluble Expression of DARPins

For further analysis, the selected clones showing specific VEGF-Axxxbinding in the crude cell extract ELISA as described above wereexpressed in E. coli XL1-blue cells and purified using their His-tagusing standard protocols. 25 ml of stationary overnight cultures (LB, 1%glucose, 100 mg/l of ampicillin; 37° C.) were used to inoculate 1 lcultures (same medium). At A(600)=0.7, the cultures were induced with0.5 mM IPTG and incubated at 37° C. for 4 h. The cultures werecentrifuged and the resulting pellets were resuspended in 40 ml ofTBS500 (50 mM Tris-HCl, 500 mM NaCl, pH 8) and sonicated. The lysate wasrecentrifuged, and glycerol (10% (v/v) final concentration) andimidazole (20 mM final concentration) were added to the resultingsupernatant. Proteins were purified over a Ni-nitrilotriacetic acidcolumn (2.5 ml column volume) according to the manufacturer'sinstructions (QIAgen, Germany). Up to 200 mg of highly soluble DARPinswith binding specificity to VEGF-Axxx could be purified from one litreof E. coli culture with a purity >95% as estimated from SDS-15% PAGE.Such purified DARPins are used for further characterizations.

Example 2: Determination of IC₅₀ Values of Selected DARPins with BindingSpecificity to VEGF-Axxx in a Spheroid Outgrowth Assay

Addition of VEGF-Axxx to HUVEC spheroids embedded in collagen matricesleads to spheroid sprouting. Addition of an inhibitor of VEGF-Axxx willblock sprout formation, which can be quantified statistically by thenumbers and lengths of sprouts. By adding different concentration ofinhibitor and a constant amount of VEGF, the IC₅₀ can be determined.

Inhibition of Spheroid Sprouting by VEGF-Axxx Specific DARPins

Spheroid outgrowth assays were done according to standard protocols(Korff et al., loc. cit.). DARPins with specificity for VEGF-Axxx wereselected and purified to >96% purity as described in Example 1. Humanumbilical vein cells were grown to confluency in monolayer culture.After trypsinization, the cell suspension was placed in a hanging dropto form spheroids, i.e. approximately 500 organized aggregated HUVECs.Spheroids were embedded in a collagen matrix and stimulated withVEGF-A165 to initiate sprout outgrowth. Sprouting inhibitors were addedadditionally to observe their effects on sprouting inhibition. Sproutnumbers per spheroid and sprout lengths were quantified using agraphical software.

The results from two example spheroid sprouting assays are shown in FIG.2 a (DARPin #30 with binding specificity for VEGF-Axxx) and FIG. 2 b(DARPin NC, a negative control DARPin with no binding specificity forVEGF-Axxx; e.g. DARPin E3_(—)5 (Binz et al., 2005, loc. cit.). The bestperforming DARPins in this assay showed IC₅₀ values in the range of 10to 50 pM, while Avastin®, Lucentis® and Macugen® showed IC₅₀ values inparallel experiments in the range of 150 and 500 pM.

Example 3: Determination of the Target Specificity of DARPin #27 inComparison to Avastin® by Surface Plasmon Resonance Analysis

Dog VEGF-A164 or Dog VEGF-A164b were immobilized in a flow cell and theinteraction of DARPin #27 (SEQ ID NO:16) and Avastin® with theimmobilized targets were analyzed.

Surface Plasmon Resonance (SPR) Analysis

SPR was measured using a ProteOn instrument (BioRad). The running bufferwas 20 mM HEPES, pH 7.4, 150 mM NaCl and 0.005% Tween 20. About 1200 RUof dog VEGF-A164 or dog VEGF-A164b were immobilized on a GLC chip(BioRad). The interactions were measured at a flow of 60 μl/min with 5min buffer flow, 100 seconds injection of Avastin® or DARPin #27 at aconcentration of 250 nM and an off-rate measurement of a few minuteswith buffer flow. The signal of an uncoated reference cell wassubtracted from the measurements.

The results are shown in FIG. 3 a (Avastin interaction with dogVEGF-A164), FIG. 3 b (Avastin interaction with dog VEGF-A164b), FIG. 3 c(DARPin #27 interaction with dog VEGF-A164) and FIG. 3 d (DARPin #27interaction with dog VEGF-A164b). Whereas Avastin clearly interacts withboth immobilized VEGF isoforms, the DARPin #27 shows only interactionwith VEGF-A164 and not VEGF-A164b.

Example 4: In Vivo Efficacy of DARPin #30 in Inhibiting VEGF-A165 in aVascular Leakage Rabbit Model

Pegylated DARPin #30 (SEQ ID NO:29) or Lucentis® is applied byintravitreal injection into an eye of a rabbit to test their efficacy toinhibit vascular leakage induced by a subsequent intravitreous injectionof human VEGF-A165.

Vascular Leakage Inhibition Measurements in Rabbits

At day 1 either PBS, PEGylated DARPin #30 (125 μg) or the equimolaramount of Lucentis® (162 μg) is applied by an intravitreal injectioninto one eye of each rabbit (treated eye). At day 4 or day 30 thetreated eye of each rabbit was challenged by intravitreal injection of500 ng of human VEGF-A165. Both eyes of all animals were evaluated 48hours after the VEGF-A165 injection by measuring the fluorescein contentin all eyes 1 h after intravenous injection of sodium fluorescein (50mg/kg animal body weight, 10%(w/v) in 0.9% (w/v)saline solution). Theratios of the amounts of fluorescence in the treated and untreated eyeswere calculated for every animal. A ratio of one corresponds to absenceof additional fluorescence leakage in the treated eye, a ratio greaterthan one indicates more fluorescence leakage in the treated eye than inthe untreated control eye.

Preparation of PEGylated DARPin

The PEGylation of protein by making use of a single Cys residue andmaleimide chemistry is well known to the person skilled in the art andcan be performed according to established protocols (e.g. from Pierce).DARPin #30 comprising an additional C-terminal linker (GGGSGGGSC, SEQ IDNO:41) was purified to near homogeneity using standard chromatographicmethods. The protein is completely reduced using DTT and purified bygel-filtration to remove the DTT and to exchange the buffer by PBS.PEG-maleimide (methoxy-poly(ethylene glycol)-oxopropylamino-propylmaleimide; NOF, no. Sunbright ME-200MA) dissolved in PBS is mixed withthe DARPin in PBS at about 15% molar excess of PEG-maleimide for 2-4hours at room temperature. The PEGylated DARPin is then separated fromnon-reactive DARPin and non-reactive PEG moieties by using standardanion exchange chromatography.

The results are shown in FIG. 4. Both PEGylated DARPin #30 and Lucentis®were able to protect the rabbit eye from VEGF-A165 induced vascularleakage 4 days after they were applied by intravitreal injections.Nevertheless, only the PEGylated DARPin #30, and not Lucentis®, was ableto protect the rabbit eye from VEGF-A165 induced vascular leakage up to30 days after the intravitreal injection.

1-20. (canceled)
 21. A recombinant binding protein comprising at leastone ankyrin repeat domain, wherein said repeat domain binds VEGF-A165with a Kd below 10⁻⁷M and wherein said repeat domain competes forbinding to VEGF-A165 with an ankyrin repeat domain selected from thegroup consisting of SEQ ID NOs:16, 22, 23, 29, 30, 33, 34, 36, 39 and40.
 22. The binding protein of claim 21, wherein said ankyrin repeatdomain competes for binding to VEGF-A165 with the ankyrin repeat domainof SEQ ID NO:29.
 23. A recombinant binding protein comprising at leastone ankyrin repeat domain, wherein said repeat domain binds VEGF-A165with a Kd below 10⁻⁷ and wherein said ankyrin repeat domain comprises anamino acid sequence that has at least 75% amino acid sequence identitywith one ankyrin repeat domain selected from the group consisting of SEQID NOs:14 to
 40. 24. The binding protein of claim 23, wherein saidankyrin repeat domain comprises an amino acid sequence that has at least80% amino acid sequence identity with one ankyrin repeat domain selectedfrom the group consisting of SEQ ID NOs:14 to
 40. 25. The bindingprotein of claim 23, wherein said ankyrin repeat domain comprises anamino acid sequence that has at least 85% amino acid sequence identitywith one ankyrin repeat domain selected from the group consisting of SEQID NOs:14 to
 40. 26. The binding protein of claim 23, wherein saidankyrin repeat domain comprises an amino acid sequence that has at least90% amino acid sequence identity with one ankyrin repeat domain selectedfrom the group consisting of SEQ ID NOs:14 to
 40. 27. The bindingprotein of claim 23, wherein said ankyrin repeat domain comprises anamino acid sequence that has at least 75% amino acid sequence identitywith the ankyrin repeat domain of SEQ ID NO:29.
 28. A pharmaceuticalcomposition comprising the binding protein of claim 21, and optionally apharmaceutical acceptable carrier and/or diluent.
 29. A pharmaceuticalcomposition comprising the binding protein of claim 23, and optionally apharmaceutical acceptable carrier and/or diluent.
 30. A nucleic acidencoding a binding protein comprising at least one ankyrin repeatdomain, wherein said repeat domain binds VEGF-A165 with a Kd below10⁻⁷M.
 31. A pharmaceutical composition comprising the nucleic acid ofclaim 30, and optionally a pharmaceutical acceptable carrier and/ordiluent.
 32. A method of treating pathological angiogenesis in a mammalincluding man, comprising administering to a patient in need thereof aneffective amount of a binding protein comprising at least one ankyrinrepeat domain, wherein said repeat domain binds VEGF-A165 with a Kdbelow 10⁻⁷M.
 33. A method of treating pathological angiogenesis in amammal including man, comprising administering to a patient in needthereof an effective amount of a nucleic acid encoding a binding proteincomprising at least one ankyrin repeat domain, wherein said repeatdomain binds VEGF-A165 with a Kd below 10⁻⁷M